Orientador: Adriana Franco Paes Leme / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T00:27:45Z (GMT). No. of bitstreams: 1 Aragao_AnnelizeZambonBarbosa1984-_D.pdf: 4629880 bytes, checksum: 7244697c557e479e2cc48f0bba48d260 (MD5) Previous issue date: 2014 / Resumo: A metaloprotease ADAM17 é uma das mais importantes reguladoras dos mecanismos de sinalização celular, pois é responsável pela liberação de ectodomínios de proteínas de superfície participando da regulação de processos fisiológicos, mas também tem sido associada à progressão de tumores e processos inflamatórios. Este estudo buscou entender a regulação da atividade proteolítica da ADAM17 por meio do seu domínio citoplasmático. Para isso, foi utilizada uma abordagem de co-imunoprecipitação seguida por análise dos parceiros de interação por espectrometria de massas. Dentre os parceiros identificados, a tioredoxina-1 (Trx-1) foi o alvo escolhido e a interação com o domínio citoplasmático da ADAM17 foi validada em experimentos complementares de microscopia confocal, ensaio de ligação em fase sólida, ressonância magnética nuclear e determinação da interface de interação por ligação química seguida de análise por espectrometria de massas. Experimentos funcionais para entender o papel funcional dessa interação foram realizados e é interessante notar que a superexpressão de Trx-1 diminui a atividade proteolítica da ADAM17 sobre o substrato HB-EGF, resultado obtido utilizando metodologia baseada em cultura de células. A descoberta de um novo parceiro de interação da ADAM17, capaz de modular a sua ativação permitirá que novos trabalhos sejam realizados para entender o mecanismo pelo qual essa metaloprotease atua / Abstract: ADAM17 is a metalloprotease which plays an important role in regulatory mechanisms of cell signaling. It is responsible for shedding of the surface proteins, participating in regulation of important physiological processes, but it has also been associated with cancer progression and inflammatory processes. The aim of this study was to explore the regulation of ADAM17 by its cytoplasmic domain. In this regard, we used an approach based on co-immunoprecipitation followed by mass spectrometry (MS). Among the identified partners, the thioredoxin-1 (Trx-1) was selected, and its interaction has been validated using confocal microscopy, solid-phase binding assay, nuclear magnetic resonance and chemical cross-linking followed by MS. To understand the functional role of this interaction, experiments using cell-based assay were performed. Trx-1 overexpression decreases HB-EGF shedding upon ADAM17 activation. The discovery of a new interaction partner of ADAM17, which can modulate its activation, gives insights for novel studies to understand the mechanism of action of this metalloprotease / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

Methylation is a post-translational modification which occurs on lysine and arginine residues. Methylation is difficult to detect due to its low abundance and lack of charge. Our laboratory previously developed a novel enrichment approach, ProMENADe, for lysine and arginine methylation in the human embryonic kidney (HEK) 293T cell line which is coupled with mass spectrometry. Simplifying a lysate with subcellular fractionation prior to enrichment increased the identification of methylation sites by 39.5% while using multiple proteases for digestion increased identification by 27%. Combining these methods yielded a 47.2% increase. Analysis at the 1% methylation level FDR filtered for C-terminal methylation identified 169 sites and further analysis revealed 74 of these sites overlap with the PhosphoSite database. This ProMENADe enrichment strategy yielded 95 novel methylation sites to the field and can be a key tool in the field of methylation allowing for the enrichment and identification of methylated proteins.

>Magister Scientiae - MSc / Autism or autism spectrum disorder (ASD) is a very diverse neurological disorder that manifests specifically in children and infants between the ages of two to three years of age. An individual suffering is deemed as autistic and individuals suffering would be classed under the banner of ASD. It is observed that sufferers have impairment in their social and interactive skills. It has both genetic and environmental factors that contribute to its diversity and although the primary cause of autism is still unclear, scientist are investigating both factors. In this study we aimed to investigate the molecular genetics of autism in the South African (SA) population. This was done in two parts, a genetic association study and afunctional genomics (proteomic study). An association study of the 2 single nucleotide polymorphisms (SNPs) of the Cadherin 10 type II gene (CDH10) (rs4307059 and rs4327572) was investigated in the SA healthy and autistic population. The proteomic approach was used to determine the differential expression of genes of the healthy population and compared to the autistic population of African descent. In both parts of the project, objectives were achieved. The SNPs were successfully genotyped however no association was determined for autism in the SA population. The urine protein profiles with 1 dimensional (1D) and 2dimensional (2D) Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis (SDSPAGE)generated in this study has revealed the following proteins, Uromodulin, Vitelline membrane outer layer protein homologue, kinninogen-1, Alpha-1-Antitrypsin, Ig Kappa chain region C, and CD59 glycoprotein that require further investigation. The results indicated that six of the identified proteins were expressed in both groups but were found to be either quantitatively or statistically significant. However, a statistically significant difference was observed in the expression of one protein (Uromodulin) which was observed to be expressed in the healthy group but absent in the experimental group. However further investigation is required validation of these findings.

The kinetics of protein phosphorylation and dephosphorylation are tightly controlled by specific kinases and phosphatases; disturbances are often disease-causing. Phosphorylation kinetics are normally monitored using radioactive isotopes of phosphorus, or by using stop-flow techniques. Approaches using mass spectrometry are severely limited by the lack of a stable isotope of phosphorus (other than 31P). The principal aim of this study is to develop a new method to incorporate 18O label into phosphorylation sites of phosphoproteins with a view of applying this method to enhance the detection of phosphorylation by mass spectrometry and to analyze the phosphorylation kinetics of proteins. Aurora-A kinase was selected to explore the possibility of using 18O-labelling to monitor phosphorylation kinetics. The kinase is well characterized, phosphorylated both in human cells and when expressed in recombinant form in E. coli and it contributes to development of some cancers when deregulated. Applying different mass spectrometric approaches resulted in the identification of 19 phosphorylation sites of Aurora-A including five new sites. Using H3P18O4 as a label donor to incorporate 18O into Aurora-A phosphorylation sites showed partial and inconsistent label incorporation. Alternatively, H218O was used to investigate the possibility of label incorporation. Preliminary results, however, showed high complex data which hampered precise identification of phosphopeptides and their labelling state. The labelling experiment was then redesigned in which induction took place in label free medium to allow the light version of the kinase to accumulate, before chasing with 18O label. This design successfully introduced fully labelled P18O3 into Aurora-A phosphorylation sites. LC ESI Q-ToF analysis of 18O labelled Aurora-A sample isolated according to this protocol identified 30 phosphopeptides showing label incorporation, which is double the number of phosphopeptides identified by MASCOT using the same MS analysis. The method was also used to investigate phosphorylation kinetics of Aurora-A. The results suggested differential regulation of phosphorylation sites of Aurora-A as some sites showed early phosphorylation while others were phosphorylated at later stages. Overall, a new approach was developed for enhanced detection of phosphorylation sites and analysis of phosphorylation kinetics.

The metabolism of absorbed xenobiotic compounds in humans results in a mixture of target compounds applicable for analysis, trapped in complex biological matrices. Gas chromatography-mass spectrometry (GC-MS) is a powerful analytical technique that has been successfully applied in the analysis of volatile and semi-volatile compounds from complex biological samples. This is due to the ability of GC-MS to separate different sample constituents at trace levels while providing accurate molecular structural information for the resolved compounds. The complexity of biological specimens and their largely aqueous nature, combined with the physicochemical properties of target analytes resulting from metabolism, greatly precludes direct analysis of biosamples by GC-MS. Traditionally, highly laborious and time consuming sample preparation procedures are performed to isolate and chemically alter target analytes to attain suitable amenity for the detection system. Furthermore, routine analytical procedures in clinical toxicology laboratories are signified by short specimen turn-around times. The commonplace use of GC-MS in modern-day laboratories still suffer from prolonged turn-around times that result from both sample preparation steps and lengthy instrumental analysis. Simplified and cost-effective analytical procedures capable of extracting multiple analytes, with divergent functional groups, from biological matrices in a timely manner are therefore required. To address this issue, this work describes the development of validated extractive-derivatization methods combined with fast GC-MS analysis for expedient and accurate quantitation of different analytes in occupational monitoring and workplace drug testing. Extractive alkylation of acidic analytes phenol, o-cresol, mandelic acid, hippuric acid, and (o-, m-, p-) methylhippuric acid for simultaneous urinary bio-monitoring of occupational exposure to benzene, toluene, ethylbenzene, and xylene, respectively, is performed. Extractive acylation for simultaneous urinary confirmation of basic analytes amphetamine, methamphetamine, norephedrine, methcathinone, ephedrine, methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) and N-methyl-1-(3,4 methylenedioxyphenyl)-2-butanamine (MBDB) in workplace drug testing is performed. The successful combination of abovementioned techniques alongside fast GC-MS allows increased sample throughput and decreased turn-around time for routine analysis while maintaining bioanalytical quantitative criteria, as required in a clinical toxicology laboratory setting. / Dissertation (MSc)--University of Pretoria, 2009. / Chemical Pathology / unrestricted

Formaldehyde cross-linking has been used to study protein-protein interactions in cells. Its short spacer arm, ability to permeate through cell membrane and the reversibility of the cross-linking reaction makes this a desirable cross-linker for in vivo studies. Although it has been widely used as a cross-linking reagent, the detailed chemistry behind protein cross-linking is not well understood. In vitro studies conducted under extended incubation periods (2 days) have shown that a multitude of amino acids are reactive to formaldehyde and that residue accessibility appears to play a role in reactivity. How applicable these findings are to formaldehyde cross-linking studies done under in vivo conditions (10-20 min incubations) is unclear. The chemistry of formaldehyde cross-linking was therefore investigated in model peptides under conditions similar to those used in in vivo studies. It was observed that only a subset of amino acids (amino termini and side chains of lysine and tryptophan) that were found reactive under extended incubation times was reactive in the much shorter incubation period. No cross-linking was detected between peptides, and elevating the peptide and formaldehyde concentrations resulted in only a minimal amount of cross-linked peptides. The relationship between residue accessibility and formaldehyde reactivity was assessed in model proteins that contain a more complex tertiary structure. It was shown that the extent of formaldehyde reactivity was dependent on the state of protein unfolding, i.e., solvent accessibility of reactive residues, and that an unfolded protein showed a significantly higher number of formaldehyde-induced modifications than a folded form, with lysine being the predominant reactive site. Formaldehyde treatment of proteins in their native form resulted in a low number of modifications even under an increased incubation time, suggesting that the protein remains folded during the course of the reaction. This is important for in vivo cross-linking studies where specificity and stability of protein-protein interactions is dictated by protein tertiary structure. / Science, Faculty of / Chemistry, Department of / Graduate

Contemporary proteomics studies require computational approaches to deal with both the complexity of the data generated, and with the volume of data produced. The amalgamation of mass spectrometry -- the analytical tool of choice in proteomics -- with the computational and statistical sciences is still recent, and several avenues of exploratory data analysis and statistical methodology remain relatively unexplored. The current study focuses on three broad analytical domains, and develops novel exploratory approaches and practical tools in each. Data transform approaches are the first explored. These methods re-frame data, allowing for the visualization and exploitation of features and trends that are not immediately evident. An exploratory approach making use of the correlation transform is developed, and is used to identify mass-shift signals in mass spectra. This approach is used to identify and map post-translational modifications on individual peptides, and to identify SILAC modification-containing spectra in a full-scale proteomic analysis. Secondly, matrix decomposition and projection approaches are explored; these use an eigen-decomposition to extract general trends from groups of related spectra. A data visualization approach is demonstrated using these techniques, capable of visualizing trends in large numbers of complex spectra, and a data compression and feature extraction technique is developed suitable for use in spectral modeling. Finally, a general machine learning approach is developed based on conditional random fields (CRFs). These models are capable of dealing with arbitrary sequence modeling tasks, similar to hidden Markov models (HMMs), but are far more robust to interdependent observational features, and do not require limiting independence assumptions to remain tractable. The theory behind this approach is developed, and a simple machine learning fragmentation model is developed to test the hypothesis that reproducible sequence-specific intensity ratios are present within the distribution of fragment ions originating from a common peptide bond breakage. After training, the model shows very good performance associating peptide sequences and fragment ion intensity information, lending strong support to the hypothesis. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Research in the field of exhaled breath analysis is developing rapidly and is currently focussed on disease diagnosis and prognosis. The ability to identify early onset of life-threatening diseases, by a subtle change in exhaled profile that is picked up through a non-invasive measure, is of clinical interest. However, implementation of exhaled breath analysis can extend further beyond disease diagnosis and/or management. Using a non-invasive and rapid sample collection with high sensitivity, breath analysis may be seen to have potential benefit to the wider community. This research describes preliminary investigations into exhaled breath in exercise-based scenarios that aims to translate current breath analysis methodologies into a sport and exercise medicine context. An adaptive absorbent-based breath sampling methodology was used to collect a total of 220 breath samples from 54 participants over 3 studies. Breath volatiles were analysed using thermal desorption-gas chromatography-mass spectrometry. Data were analysed with targeted, and multivariate metabolomics-based approaches. Potential health impacts to high performance and recreational swimmers exposed to chlorinated water was studied. Following preliminary and scoping studies, 19 participants were sampled before a 30 min swim, and a further 5 times for 10 hrs after swimming. Environmental and control samples were also collected. Concentrations of chlorine-based disinfection by-products were observed to increase by up to a median of 121-fold, and take up to 8.5 hrs to return to pre-swimming levels. Metabolomic profiling identified the monoterpene geranylacetone to be a discriminant variable in samples taken 10 hrs after swimming. Geranylacetone is associated with membranes and extracellular fluids and an upregulated trend was observed across the five sampling time points post-swimming. Further research with an appropriately stratified and powered cohort (n=38) was recommended. The effects of intense exercise on breath profiles was explored for the possible use of breath analysis for exercise science with elite performance-based medicine. Twenty-nine participants provided exhaled breath samples before undergoing a maximal oxygen uptake (fitness) test and then provided 2 additional samples over the following 1 hr period. High and low fitness groupings, deemed by oxygen uptake values, were compared for exhaled metabolites. Lower exhaled acetone and isoprene were observed in participants with greater absolute oxygen uptake leading to a hypothesis for a non-invasive breath based fitness test. Finally, an interface for breath-by-breath analysis using a transportable mass spectrometer was developed. A controlled change in exhaled profiles was achieved through the ingestion of a peppermint oil capsule. Menthone was measured on-line and monitored for up to 10 hrs post-administration. Sixteen participants enabled the system to be demonstrated as exhaled menthone was at elevated concentrations for at least 6 hrs. Validation against thermal desorption-gas chromatography-mass spectrometry confirmed the system to be detecting metabolites at the sub-μg L-1 range.

This thesis is concerned with the problems of sample preparation arising in the study of lead isotope abundances. The importance of this study to geophysics has been amply shown by R.D. Russell, R.M. Farquhar, F.G. Houtermans, J.T. Wilson, H.F. Ehrenberg and many others. Chapter 1 gives an outline of lead isotope measurement techniques, including types of mass spectrometers generally used and some of the problems encountered. The mass spectrometer used in the present research was designed and constructed by R.D. Russell and F. Kollar and descriptions of it will be found in their publications and in F. Kollar's Ph.D. thesis. The present techniques of producing lead tetramethyl for isotopic analysis from ore samples are discussed in Chapter 2. The remaining chapters deal with the purification of lead tetramethyl for mass spectrometer analysis, using vapour phase chromatography. This technique has found immediate application in the precise intercomparison of lead samples recently carried out in the Geophysics Laboratory at the University of British Columbia by F. Kollar and others (F. Kollar, R.D. Russell and T.J. Ulrych, in press). The long range object for developing this technique is to purify lead tetramethyl prepared by free methyl radicals reacting with metallic lead (cf. A.J. Surkan 1956) prior to isotopic analysis. The presence of impurities in samples prepared this way has discouraged the development of this method in the past. The final chapter deals with this aspect of the proposed problem. This thesis is intended as a preliminary to the writer's Ph.D. research which will also deal with isotopic lead analysis. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

Anionic metal-oxalate complexes have been generated in the gas phase and an attempt at determining plausible structures were made. Two different experimental techniques were coupled to mass spectrometry: Infrared Multiphoton Dissociation (IRMPD) and ion mobility. Both techniques were compared to theoretical structures calculated using various levels of theory. With the use of IRMPD, frequencies were generated for each complex and compared to theoretical frequencies. Plausible structures for all complexes were found using the M-series of density functional levels of the theory when the 6-311+gd basis set was used and Bhandhlyp functional was appropriate for the lanl2dz basis set. Using ion mobility allowed for collision cross-sections to be calculated and compared to theoretical collision cross-sections of the various structures. Unfortunately no plausible structures were determined using this technique due to a lack of calibrants for the negative mode of ion mobility.